title: Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting.
abstract: Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action.
authors.affiliation: Single Molecule Analysis in Real-Time (SMART) Center, University of Michigan, Ann Arbor, MI 48109-1055, USA.
authors.forename: Sethuramasundaram
authors.initials: S
authors.lastname: Pitchiaya
authors.forename: Vishalakshi
authors.initials: V
authors.lastname: Krishnan
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Custer
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D000818:Animals; D001483:Base Sequence; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D012261:Ribonucleoproteins / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D035683:MicroRNAs; D012261:Ribonucleoproteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
keywords: Non-coding RNA; RNA silencing; Single molecule microscopy; Single particle tracking; microRNA